OF PROPOSAL Al 27690 YEARS 16-20 "Structures of HIVreverse transcriptase with substrates" Overall Aim: Determine structures of HIVRTthat will aid development of novel anti-AIDS drugs and enhance our understanding of RTfunction and mechanisms of catalysis, drug inhibition, and drug resistance HIV reverse transcriptase (RT) is the target of many key anti-AIDS drugs. Nucleoside, nucleotide, and non-nucleoside RT inhibitors are used as effective drugs for treating AIDS, but success can be limited by the emergence of drug-resistant viral variants. Promising non- nucleoside RT inhibitors that inhibit all common drug-resistant mutants have been developed using structure-based methods and we propose to carry out crystallographic studies that will enable structure-based improvement of additional classes of RT inhibitors targeting multiple sites on the enzyme. The use of strategically modified proteins and nucleic acids will enhance the quality and incisiveness of the structural and mechanistic studies. The proposed structural studies are also designed to enhance our understanding of basic mechanisms of inhibition and drug resistance. The proposed work relies on collaborations with: 1) Dr. Stephen Hughes (NIH NCI- Frederick) for production of wild-type and mutant RT including enzymeswith strategically placed cysteine residues, and for carrying out parallel biochemical and genetic studies of HIV-1 RT; 2) Dr. Donald M Jerina (NIDDK) and 3) Dr. Roger Jones (Rutgers University) for synthesis of tailored nucleic acid reagents for structural and mechanistic studies. The studies proposed here are entirely complementary to structural studies of HIV-1 RT funded on other grants to our laboratory (P01 GM 66671, Janssen Research Foundation, Wyeth Pharmaceuticals). Now that we have obtained many useful types of RT crystals, relevant to multiple classes of inhibitors, our capacity to determine new RT structures is directly proportional to the personnel effort that we can commit to these studies.